The long-term objective of this project is to understand how a eukaryotic chromosome replicates and segregates. To this end a 200 kilobase (kb) circular derivative of Saccharomyces cerevisiae chromosome III that includes 60% of the chromosome has been cloned and restriction mapped. This chromosome is being used to study location, structure, and pattern of use of replication origins and to identify genes whose products are necessary for the replication of this chromosome. This proposal addresses three issues related to the replication of this chromosome. First, replication origins used in vivo are being mapped using 2D gel techniques, and positions of origins are being compared with known positions of ARS elements, which promote autonomous replication of plasmids. Replication origins will be deleted and moved to determine how many are required for normal chromosome stability and whether the temporal patterns of replication or the pattern of origin usage is perturbed by changes in origin spacing or changes in chromosome context. Second, the DNA sequences required for origin function will be further defined by isolating additional mutant origins using in vitro mutagenesis techniques and characterizing their function both in plasmids and in their normal chromosomal context. Third, proteins that interact with ARS elements will be identified using both novel genetic screens and biochemical approaches. These studies should greatly increase our understanding of a fundamental cellular process, DNA replication. These questions cannot be approached in higher eukaryotes at the present time because their chromosomal DNA's are too large to isolate without breakage and because no autonomously replicating chromosomal sequences have been identified which function in the cells from which they were isolated. Knowledge we gain from the yeast system should be applicable to higher cells, and may help lead to an understanding of the basis of unscheduled DNA synthesis, which is characteristic of malignant cells.